Understanding abnormal embryonic development is critical for determining the mechanistic processes involved in xenobiotic-induced human birth defects. Although it has been difficult to pinpoint xenobiotic-defect relationships, the potential of many environmental agents to elicit human birth defects has been well documented. This body of data includes the suggestions that compounds such as toluene and methyl parathion are associated with the induction of limb defects. The molecular mechanisms by which environmental agents exert their effects are unknown. It is known, however, that the alterations in specific developmentally relevant genes are associated with a number of birth defects. Chemical agents can disturb normal developmental processes by altering gene expression. 5-aza-2U-deoxycytidine (d-AZA) is a cytidine analog and a potent DNA demethylating agent. Changes in gene expression are strongly associated with changes in DNA methylation patterns during development. Recently the investigators have shown that d-AZA elicits hindlimb specific defects in vivo when the compound is administered to pregnant mice on day 10 of gestation. It is therefore hypothesized that d-AZA induces hindlimb defects via inappropriate gene expression subsequent to DNA demethylation. In order to test this hypothesis the investigators propose the following: 1) to determine the effects of d-AZA on expression (via RT-PCR) of genes already known to be associate with limb patterning; 2) to completely characterize the candidate cDNA clones previously isolated via subtractive hybridization studies subsequent to d-AZA treatment; 3) to determine changes in the methylation status (via isoschizomer studies) of the genomic equivalent of the isolated cDNA clones subsequent to d-AZA treatment; and 4) to determine the temporal and anatomical expression pattern of the isolated clones as well as known genes associated with limb patterning. This model system will have relevance both to the induction of limb (and other skeletal) defects by chemicals found in the environment, and to the basic understanding of the developmental processes involved in limb formation.